ABSTRACT Malaria is a major public health burden in many tropical regions of the world, causing a half million deaths and two hundred million clinical episodes every year. The utility of the first-line anti-malarial regimen, artemisinin combination therapy (ACT), is threatened by emerging resistance. Development of mechanistically distinct anti-malarials will be necessary to replace ACT once parasite resistance inevitably becomes widespread. Understanding fundamental biochemical processes that underpin both asexual and sexual parasite stages in the human host erythrocyte will stimulate the discovery of new drugs to treat disease and block transmission. This proposal seeks to accelerate the discovery and functional characterization of lipases, enzymes that catalyze the hydrolysis of a large, diverse class of biological molecules termed lipids. In Aim 1, a serine hydrolase activity-based probe will be employed to affinity-purify putative parasite lipases, which will then be identified using mass spectrometry. In a complementary approach, two high molecular weight putative lipases will be epitope tagged to assess their contributions to lipolytic transformations in the asexual malaria parasite. To establish the importance of the lipases uncovered in the discovery phase, the genes encoding the enzymes will be targeted for gene disruption. In Aim 2, the discovery of lipases in early and mature gametocytes will be accomplished using the activity-based probe/inhibitor competition approach that has proven successful in asexual parasites. These studies will provide a picture of the diversity of lipolytic serine hydrolases in asexual parasites and in developing and mature gametocytes. They will reveal critical and potentially druggable enzymes and may lead to the discovery of novel biochemical processes that allow the malaria parasite to flourish in human erythrocytes.